In Situ Quantification of Lipids in Live Cells by Using Lipid-Binding Domain-Based Biosensors.

Lipid molecules contribute to a large extent to the regulation of cellular signaling, as cellular signals are generated primarily through the selective interaction of various cellular proteins with lipids in the plasma membrane. Hence the location, concentration, and duration of lipids on the cell membrane are critical for the selection of proteins and the initiation of signaling. To monitor the concentration and location of lipid molecules on the cell membrane, researchers have developed a variety of lipid biosensors that allow quantitative in situ visualization of lipid molecules in living cells based on lipid-binding domains with high specificity, sensitivity, and biocompatibility, providing a powerful tool for the study of cellular signaling mechanisms involving lipid molecules. In this review, we first introduced the emergence of lipid-binding domains and then focused on the practical considerations on how to implement the lipid sensor, including probe selection, modification, characterization, and imaging techniques. We then described experimental observables and the relevant physicochemical parameters in the context of single-molecule studies in cells. Finally, we presented our views on the future development of lipid sensors and methods for lipid quantification.

The renoprotective effect of curcumin against cisplatin-induced acute kidney injury in mice: involvement of miR-181a/PTEN axis.

Background: Nephrotoxicity, especially acute kidney injury (AKI), is the main dose-limiting toxicity of cisplatin. Although recent studies showed that curcumin prevented cisplatin-induced AKI effectively, further studies to understand the mechanism are required.Methods: We established an AKI mouse model. Male C57BL/6 mice were assigned to three groups: saline group (control), cisplatin group (CP), and curcumin + cisplatin group (CP + Cur). The CP group received a single intraperitoneal (i.p.) injection of cisplatin, while the control group received saline. The CP + Cur group received i.p. curcumin three days before cisplatin injection and curcumin administered for another three days until the day before euthanization. Renal injury was assessed by serological and histological analysis. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the phosphatase and tensin homolog (PTEN), and microRNA (miR)-181a expression in the renal tissues. Bioinformatics prediction and western blotting methods validated the targets of miR-181a in vitro.Results: Curcumin treatment alleviated cisplatin-induced nephrotoxicity as validated by the blood urea nitrogen (BUN) values, and histological analysis of kidneys. At the molecular level, curcumin treatment decreased miR-181a expression level, which was induced by cisplatin and restored the in vivo expression of PTEN, which was suppressed by cisplatin. We verified the direct regulation of PTEN by miR-181a in cultured human embryonic kidney 293T cells.Conclusions: We showed the involvement of miR-181a/PTEN axis in the renoprotective effect of curcumin against cisplatin-induced AKI, and provide new evidence on the ability of curcumin to alleviate cisplatin-induced nephrotoxicity.

[Construction of Vectors Expressing Inhibiting Peptides for Nuclear Import and Its Effect on Growth and Migration of HeLa Cell].

OBJECTIVE: To construct the expression vectors for red fluorescent protein fused with inhibiting peptides for nuclear import (Bimax),and explore the location of Bimax and its potential effects on cell proliferation and migration in HeLa cells. METHODS: Two kinds of polynucleotide encoding inhibiting peptides for nuclear import were synthesis respectively and subsequently annealed for inserting into vector pDs-Red-C1. The recombinant plasmids were transfected into competent bacterial DH-5α. After transfection,the positive bacteria were picked up for DNA sequencing. The recombinant plasmids pDs-Red-Bimax2,pDs-Red-Bimax1 and negative plasmid pDs-Red-C1 were transfected into HeLa cells respectively according to Lipofectamine2000 protocol. After transfection,the expression and location of red fluorescent protein were observed with fluorescence microscope. Furthermore,MTT assay and cell-migration assay were used to detect the proliferation and migration of Bimax transducted cells. RESULTS: DNA sequencing showed that the polynucleotides encoding Bimax1 or Bimax2 were inserted into pDs-Red-C1 vector successfully. After transfected into HeLa cells,the inhibiting peptide induced red fluorescent protein locating in nuclear. Furthermore,either the fusion protein RFP-Bimax1 or RFP-Bimax2 can suppress the proliferation and migration of HeLa cells. CONCLUSION: The expression vectors for red fluorescent protein fused with inhibiting peptides for nuclear import were successfully constructed. In addition,the fusion proteins were expressed and located in nuclear and suppressed the proliferation and migration of tumor cells.

Investigation on reference intervals and regional differences of platelet indices in healthy Chinese Han adults.

BACKGROUND: Reference intervals are important for interpretation of clinical laboratory tests. The platelet (PLT) indices such as the mean platelet volume (MPV) and platelet distribution width (PDW) are newer hematological parameters, which have been recently reported as clinically valuable biomarkers. However, there are not many studies that have estimated the reference intervals for these parameters in healthy Chinese Han adults. OBJECTIVE: The objectives of this study were to establish reference values of PLT indices [including PLT count, MPV, PDW, platelet-large cell ratio (P-LCR), and plateletcrit (Pct)] for healthy Chinese Han adults. We also aimed to determine the region-based differences of PLT indices in China. METHODS: A total of 4,642 volunteers with a mean age of 43 were recruited from six regions of China. PLT indices were performed on Sysmex XE-2100 hematology analyzers, whose traceability was well verified. RESULTS: There were significant region-based differences for all PLT indices. Reference people in Chengdu had the lowest mean PLT count and Pct, but the highest MPV, PDW, and P-LCR among the six regions. Therefore, we derived the reference intervals in Chinese Han population excluding Chengdu reference people for PLT indices as PLT count: (127-341) × 10(9)/l; MPV: (9.20-13.30) fl; PDW: 9.90-19.00%; P-LCR: 18.10-52.00%; Pct: 16.00-41.00%. CONCLUSIONS: Region-specific reference intervals are essential as there were statistically significant region-related differences in the PLT parameters. The reference intervals established in this study differed from the existing reference values. Chengdu region may need proper specific reference ranges, which apply to their people, for all PLT parameters.

A simple point-of-care microfluidic immunomagnetic fluorescence assay for pathogens.

In this work, we reported a simple rapid and point-of-care magnetic immunofluorescence assay for avian influenza virus (AIV) and developed a portable experimental setup equipped with an optical fiber spectrometer and a microfluidic device. We achieved the integration of immunomagnetic target capture, concentration, and fluorescence detection in the microfluidic chip. By optimizing flow rate and incubation time, we could get a limit of detection low up to 3.7 × 10(4) copy/µL with a sample consumption of 2 µL and a total assay time of less than 55 min. This approach had proved to possess high portability, fast analysis, high specificity, high precision, and reproducibility with an intra-assay variability of 2.87% and an interassay variability of 4.36%. As a whole, this microfluidic system may provide a powerful platform for the rapid detection of AIV and may be extended for detection of other viral pathogens; in addition, this portable experimental setup enables the development of point-of-care diagnostic systems while retaining adequate sensitivity.

In-situ quantification of lipids in live cells through imaging approaches.

Lipids are important molecules that are widely distributed within the cell, and they play a crucial role in several biological processes such as cell membrane formation, signaling, cell motility and division. Monitoring the spatiotemporal dynamics of cellular lipids in real-time and quantifying their concentrations in situ is crucial since the local concentration of lipids initiates various signaling pathways that regulate cellular processes. In this review, we first introduced the historical background of lipid quantification methods. We then delve into the current state of the art of in situ lipid quantification, including the establishment and utility of fluorescence imaging techniques based on sensors of lipid-binding domains labeled with organic dyes or fluorescent proteins, and Raman and magnetic resonance imaging (MRI) techniques that do not require lipid labeling. Next, we highlighted the biological applications of live-cell lipid quantification techniques in the study of in situ lipid distribution, lipid transformation, and lipid-mediated signaling pathways. Finally, we discussed the technical challenges and prospects for the development of lipid quantification in live cells, with the aim of promoting the development of in situ lipid quantification in live cells, which may have a profound impact on the biological and medical fields.

MiR-186 bidirectionally regulates cisplatin sensitivity of ovarian cancer cells via suppressing targets PIK3R3 and PTEN and upregulating APAF1 expression.

Ovarian cancer is a highly lethal malignancy in the female reproductive system. Platinum drugs, represented by cisplatin, are the first-line chemotherapeutic agents for treatment of various malignancies including ovarian cancer, but drug resistance leads to chemotherapy failure. MicroRNAs emerged as promising molecules in reversal of cisplatin resistance. MiR-186 was reported to be downregulated in the cisplatin-resistant ovarian cell lines and miR-186 expression increased cisplatin sensitivity. However, we found the bidirectional regulatory effects of miR-186 on cisplatin sensitivity for the first time that overexpression of miR-186 at low concentration increased the cisplatin sensitivity of ovarian cancer cells A2780/DDP, while high concentration of miR-186 decreased the cisplatin sensitivity. The survival assay in other types of cancer cell lines verified the bidirectional regulatory function of miR-186 on cisplatin sensitivity in dose and cell type dependent manners. MiR-186 suppressed the protein levels of PTEN and PIK3R3 dose-dependently, which are opposite regulatory molecules of the oncogenic AKT pathway. MiR-186 also enhanced the protein levels of apoptotic gene APAF1 dose-dependently. We proposed the final effects of PTEN and APAF1 outweighed PIK3R3 when miR-186 at low concentration so as to increase the cisplatin sensitivity of ovarian cancer cells, while the final effects of PIK3R3 outweighed PTEN and APAF1 when miR-186 at high concentration so as to decrease the cisplatin sensitivity. We concluded the outcome of regulation of these opposite functional molecules contributed to the bidirectional regulatory effects of miR-186 in ovarian cancer cisplatin sensitivity. It deserves more attentions when developing therapeutic strategies based on the bidirectional functional miRNAs.

Rapid detection and subtyping of multiple influenza viruses on a microfluidic chip integrated with controllable micro-magnetic field.

Influenza viruses have threatened animals and public health systems continuously. Moreover, there are many subtypes of influenza viruses, which have brought great difficulties to the classification of influenza viruses during any influenza outbreak. So it is crucial to develop a rapid and accurate method for detecting and subtyping influenza viruses. In this work, we reported a rapid method for simultaneously detecting and subtyping multiple influenza viruses (H1N1, H3N2 and H9N2) based on nucleic acid hybridization on a microfluidic chip integrated with controllable micro-magnetic field. H1N1, H3N2 and H9N2 could be simultaneously detected in 80min with detection limits about 0.21nM, 0.16nM, 0.12nM in order. Moreover, the sample and reagent consumption was as low as only 3µL. The results indicated that this approach possessed fast analysis and high specificity. Therefore, it is expected to be used to simultaneously subtype and detect multiple targets, and may provide a powerful technique platform for the rapid detection and subtyping analysis of influenza viruses.

Acupuncture through Baihui (DU20) to Qubin (GB7) mitigates neurological impairment after intracerebral hemorrhage.

Inflammation plays an important role in nerve defects caused by intracerebral hemorrhage. Repairing brain damage by inhibiting the macrophage-inducible C-type lectin/spleen tyrosine kinase (Mincle/Syk) signaling pathway is a potential new target for treating cerebral hemorrhage. In this study, we aimed to determine whether acupuncture through Baihui (DU20) to Qubin (GB7) is an effective treatment for intracerebral hemorrhage through the Mincle/Syk signaling pathway. An intracerebral hemorrhage rat model was established by autologous blood infusion into the caudate nucleus. Acupuncture through Baihui to Qubin was performed for 30 minutes, once every 12 hours, for a total of three times. Piceatannol (34.62 mg/kg), a Syk inhibitor, was intraperitoneally injected as a control. Modified neurological severity score was used to assess neurological function. Brain water content was measured. Immunohistochemistry and western blot assay were used to detect immunoreactivity and protein expression levels of Mincle, Syk, and CARD9. Real-time polymerase chain reaction was used to determine interleukin-1ß mRNA levels. Hematoxylin-eosin staining was performed to observe histopathological changes. Our results showed that acupuncture through Baihui to Qubin remarkably improved neurological function and brain water content, and inhibited immunoreactivity and expression of Mincle, Syk, CARD9, and interkeukin-1ß. Moreover, this effect was similar to piceatannol. These findings suggest that acupuncture through Baihui to Qubin can improve neurological impairment after cerebral hemorrhage by inhibiting the Mincle/Syk signaling pathway.

[Clinical observation on treatment of severe acute pancreatitis by combined administration of Qingyi Decoction and Glauber's salt].

OBJECTIVE: To investigate the value of Qingyi Decoction (QYD) and Glauber's salt (GS) for treatment of severe acute pancreatitis (SAP). METHODS: Sixty-five patients with SAP were randomly assigned to two groups, the treated group (33 patients) and the control group (32 patients). They received the same therapy except that to the patients in the treated group, QYD was given through naso-gastric tube and GS was applied externally. RESULTS: Compared with those in the control group, the alleviating time of clinical symptoms, such as abdominal pain and distention, time of hospitalization and time of blood amylase recovery in the treated group were shorter, with lesser complications and lower rate of transferring to operation (all with P < 0.05), showing a better efficacy in all aspects, but the mortality in the two groups was not different (P > 0.05). CONCLUSION: Combined use of QYD and GS has significant therapeutic effect for treatment of SAP.

[Expression of miR-140-5p and prediction of its target gene in human mesenchymal stem cells during adipogenic differentiation].

OBJECTIVE: To screen the differentially expressed miRNAs and their target genes in adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) to better understand the mechanism for regulating the balance between osteoblast and adipocyte differentiation. METHODS: Cultured hMSCs were induced for adipogenic differentiation, and at 0, 7, 14, and 21 days of induction, the cells were examined for miRNA and mRNA expression profiles using miRNA chip and transcriptome sequencing (RNA-seq) techniques. Correlation analysis was carried out for the miRNAs and mRNAs of potential interest. The databases including TargetScan, PicTar and miRanda were used to predict the target genes of the differentially expressed miRNA. RESULTS: The expression of miR-140-5p was down-regulated and leukemia inhibitory factor receptor (LIFR) expression increased progressively during adipogenic differentiation of hMSCs, showing a negative correlation between them. Target gene prediction using the 3 databases identified LIFR as the target gene of miR-140-5p. CONCLUSION: miRNA-140-5p may play an important role by regulating its target gene LIFR during adipogenic differentiation of hMSCs.

Synthesis of N-acylamidines via rhodium-catalyzed reaction of nitrosobenzene derivatives with N-sulfonyl-1,2,3-triazoles.

α-Imino rhodium carbene, readily generated from N-sulfonyl-1,2,3-triazole, underwent cycloaddition and subsequent rearrangement with a nitrosobenzene derivative to afford N-acylamidine. The unprecedented C-C bond cleavage of α-imino carbene was facilitated by the weakness of the N-O bond.

Synthesis of 3-pyrrolin-2-ones by rhodium-catalyzed transannulation of 1-sulfonyl-1,2,3-triazole with ketene silyl acetal.

α-Imino rhodium carbenoids generated from 1-sulfonyl 1,2,3-triazole were applied to the 3 + 2 cycloaddition with ketene silyl acetal, offering a novel and straightforward synthesis of biologically interesting compound 3-pyrrolin-2-one with broad substrate scope.

Synthesis of 3-aza-bicyclo[3.1.0]hexan-2-one derivatives via gold-catalyzed oxidative cyclopropanation of N-allylynamides.

N-allylynamides with various functional groups and different substitution patterns can be converted into 3-aza-bicyclo[3.1.0]hexan-2-one derivatives in moderate to high yield using IMesAuCl/AgBF4 as the catalyst and pyridine N-oxide as the oxidant. A noncarbene mediated approach is proposed as the mechanism.